Project Summary/Abstract Electron paramagnetic resonance (EPR) spectroscopy is a critically important technique in biomedical research with a unique ability to detect naturally occurring or engineered unpaired electrons in complex biological environments. EPR has wide-ranging applicability to structural biology, metalloprotein research, redox biology, rational drug design, and clinical diagnostics. Groundbreaking advancements in sample volume requirements for biomedical EPR spectroscopy applications were made nearly four decades ago with the development of the loop-gap resonator (LGR), which represented breakthrough benefits such as 10-fold lower sample volume requirements and higher resonator efficiencies to increase signals over the commonly used cavity resonator. To accommodate the biomedical needs of even further increased signal intensity and to provide the broader scientific community with accessible technology, we propose to capitalize on our recent development of the dielectric LGR (dLGR) concept and optimize this resonator technology to increase sensitivity beyond that achieved upon introduction of the LGR. A dLGR is effectively a small dielectric resonator placed inside the inner loop of an LGR where the return flux of the dielectric flows through the outer loops of the LGR. Analytic theory and our high-frequency structure simulations indicate that the dLGR enables an order-of-magnitude improvement in sensitivity over the LGR. To take full advantage of the extremely low volume requirements for the dLGR, we propose to develop efficient sample handling technologies that couple the dLGR to high- throughput sample handling instrumentation. We aim to develop two transformative technologies for biomedical EPR applications: i) a nano-dLGR with a 10-fold increase in sensitivity for ~0.2 µL sample volumes integrated with a customized autosampler and ii) a micro-dLGR for a dramatic increase in sensitivity for ~2 µL sample volumes with an optimized stopped-flow system for millisecond time scale kinetics measurements. These transformative and innovative prototypes with outstanding sensitivity will be easy to use and ultimately widely available to the scientific community. Where the LGR was a transformative advance compared with cavity resonators, the dLGR is expected to be another transformative leap from an LGR.

Project Narrative Electron paramagnetic resonance (EPR) spectroscopy is a critically important technique in biomedical research with a unique ability to detect naturally occurring or engineered unpaired electrons in complex biological environments. We will develop two innovative EPR spectrometer technologies with outstanding sample sensitivity that are easy to use and widely available to the scientific community. The resulting state-of-the-art prototypes will provide a transformative increase in throughput that will enable a wide range of new applications in biomedical EPR spectroscopy studies including structural biology, metalloprotein research, redox biology, rational drug design, and clinical diagnostics for a range of disease areas.